The project deals with the early events in HIV-1 replication. Initially the sequence requirements for the initiation of (+) strand DNA in HIV-1 will be studied. A viral system will be developed to allow the replacement of the wild-type Polypurine Tract (WT PPT) with specific mutations, which have been determined using an in vitro assay. The sequence requirements will further be defined by doing an "in vivo selection" assay. This basically entails replacing the WT PPT with a pool of mutations made in vitro. The mutations will then be replaced into a NL4-3 virus background and allowed to replicate. Through successive rounds of replication and re-infection the viruses, which can best replicate, will be selected. The RNA from the selected viruses will be extracted and RT-PCR performed. Finally, pools of virus and individual clones of the RT-PCR products will be sequenced to determine those sequences, which can support replication. The same strategy will also be used to determine the sequence requirements of a region immediately upstream of the PPT known as the U-box region. Together this information will provide detailed information on the reverse transcription step in viral replication.